De Cuyper M., Bulte J.W.M. - Physics and chemistry basis of biotechnology (Vol. 7) (2002)(en)

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enzyme in the presence of the same molecules on the spectrophotometer. In all cases, hydrogen peroxide formation due to uncoupling was minimal.

Figure 12. Structure of the pesticides and PAHs screenedfor turnover by random mutants of P450 BM3.

Similarly, the same assay was applied to investigate the interaction of various compounds, including a number of polycyclic aromatic hydrocarbons (PAHs) and pesticides (Figure 12), against a library of P450 BM3 variants, with random mutations in the haem domain region. Two variants have been identified among about 320 screened, showing a significantly different pattern of turnover of certain of the compounds compared to the wild type. Characterisation of the two active mutants is in progress.

4. Designing a human/bacterial2El-BM3 P450 enzyme

P450 2E1 is a microsomal P450 present in the liver and other tissues of many mammalian species that has been shown to catalyse the oxidation of over 80 compounds, including benzene, ethanol, acetone, chloroform, many nitrogenous compounds together with drugs such as acetaminophen and chlorzoxazone. Having as substrates ethanol and many suspect carcinogens, P450 2E1 has been considered of great interest for its possible relevance to alcoholism, chemical carcinogenesis and other diseases [84]. Its substrates have diverse structures but most of them have the common characteristic of being low molecular weight molecules [ 146].

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In the absence of P450 2E1 crystal structure its active site conformation is not known. However, studies on its different substrates and competitive inhibitors, have provided useful information on some of the properties of its active site including:

•it binds and efficiently oxidises small molecules,

•accommodates water-soluble molecules although hydrophobicity is an important feature of the substrate-binding pocket,

•does not effectively accommodate molecules with a formal ionic charge [ 146].

This section reports on the modelling, construction and expression of a chimeric protein designed to confer the human P450 2E1 new and improved properties, like solubility and self-sufficiency in catalysis, hence conferring the ability of receiving electrons directly from small electron donors (NADPH), maintaining, at the same time, its quite peculiar substrate specificity. Self-sufficient and soluble cytochrome P450 systems are, in fact, essential to employ the catalytic power of these enzymes for biotechnological purposes.

The chimeric protein was obtained by fusing part of the human P450 2E1 with a portion of P450 BM3. This latter cytochrome exhibits key features, as mentioned earlier, that make it an ideal candidate for fusion with the human P450 2E1:

•it is catalytically self-sufficient due to the presence of a reductase domain within the same polypeptide chain, containing both FMN and FAD and requiring only NADPH for activity,

•it is highly homologous to the human P450 enzymes (30% homology between the P450 BM3 haem domain and 2E1), and it shares many common features leading to the classification in the same class II as the human microsomal P450

enzymes.

Furthermore, the three-dimensional structure of P450 BM3 has been solved [46, 147], its gene has been cloned [66] and its catalytic properties have been studied.

4.1. MODELLING

Previous available models of mammalian P450s were often approximate since they were based either solely on the structure of P450cam, that has a very low sequence identity with mammalian enzymes (from ca 15 to 20%) [148], or on models of the binding site which is expected to be the most variable region of the protein. Only recently, improved models were obtained after observing the necessity to generate a multiple sequence alignment of the target P450 with template proteins. These alignments should be done by looking at the structurally conserved regions of the templates and not simply relying on automated alignment procedures, since these are often not reliable, especially for some regions of the protein [148].

To help the design of a new valid chimera likely to possess the desired properties, a three-dimensional model of P450 2E1 was built in this laboratory. The P450 2E1 model was generated using a Silicon Graphics Indigo2 IRIX 6.2 workstation equipped with the Biosym/MSI software Insight. The following protocol was used for the designing of the model:

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•four related proteins of known X-ray structure were chosen (P450terp, P450cam, P450eryF and the BMP domain of P450 BM3) and their sequences were aligned with P450 2E1 using the structurally conserved regions (SCRs),

•the co-ordinates of the structurally variable regions (VRs) were assigned using different templates for different VRs, depending on the degree of homology (when the VRs were of different length the co-ordinates were assigned by a loop search),

•a possible initial conformation of the side chains was searched and the coordinates of the more flexible N- and C- terminal regions were arbitrarily assigned,

•incorrect steric contacts ( bumps ) were corrected by manually orienting the involved rotamers, and finally

•the model was refined and energy minimised. A model of the P450 2E1-BM3 was obtained using the same procedure.

4.2. CONSTRUCTION

The information gained from the preliminary model of CYP 2E1 and previous works on isozymes [88, 149-1521, was used to design a chimeric cytochrome P450,2El-BM3. This chimeric P450 contained the first 54 residues at the N-terminal of P450 BM3 (fragment I), the whole sequence of P450 2E1 from residue 81 to the C-terminal (fragment II) and the whole reductase domain of P450 BM3 (fragment III). The three fragments were successfully isolated from the respective cloned genes and compatible restriction sites were inserted, by PCR, at their extremities using different mutagenic oligos. The whole construct of 3150 base pairs, containing the three fragments together, was cloned back into the pT7Bm3HdZ vector [66] for the inducible expression in E. coli.

4.3. EXPRESSION AND FUNCTIONALITY

The 2E1-BM3 chimera with a molecular weight of 118 kDa was efficiently expressed. When reduced by sodium dithionite in a carbon monoxide saturated atmosphere the characteristic 450 nm peak was observed, giving support to a folded and functional chimeric protein.

TG test the activity of the 2E1-BM3 chimeric enzyme in whole cell lysates the screening assay described earlier was used. The oxidation of NADPH by P450 2E1BM3 in the presence of two known P450 2E1 substrates (ethanol and arachidonate) and a known 2E1 inhibitor (isoniazid) was investigated. Lysates of cells expressing P450 BM3 and non-transformed cells were used as controls. The results are shown in Figure 13, where absorbance ratio is the ratio of the NADP+-alkali product given by cells expressing the P450 2E1-BM3 chimera or the control P450 BM3, against that given by non-transformed cells.

Overall these results show how the newly engineered bacterial/human P450 enzyme is active. Moreover the power of the screening method developed in this laboratory is demonstrated in its ability to identify positive, active enzymes in whole cells.

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Compound

Figure 13 Screening of P450 2EI-BM3 (white) using ethanol (A), isoniazid (B) and arachidonic acid (C) at increasing concentrations from left to right Cytochrome P450 BM3 was used as a control (black)

5. Conclusions

This work has shown how protein engineering methodologies can provide many useful extensions in the use of defined molecules for biosensing purposes. The experimentalist is no longer limited by the properties of the natural proteins/enzymes.

Electrochemical contact with the electrode was enhanced in the artificial chimera between flavodoxin and the haem domain of P450 BM3 (fld-BMP). Moreover, the availability of an assay able to screen for

•NAD(P)H-linked enzymatic activity towards molecules of pharmacological (new potential drugs) and biotechnological (bioremediation and biosensing) interest,

•libraries of random mutants of NAD(P)H-dependent enzymes with desired

catalytic specificities,

opens many new possibilities in the engineering of novel P450 enzymes. A successful application of the assay as a method for identifying positive, active enzymes was demonstrated for the P450 2El-BM3 chimera.

The identification of a number of active variants of P450 BM3 against several pollutants which will be used for the creation of chimeric P450 arrays with desired specificities, will follow. Finally, this approach could be seen as a step forward towards

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“fourth generation” of biosensors which will be based on artificial redox proteins with predicted ET pathways.

Acknowledgements

The authors wish to thank the following funding bodies for their support; EC biotechnology programme CT960413 (S.J.S.), the National Scholarship Foundation of Greece (G.E.T.) and BBSRC studentship 99/B1/E/05953 (M.F.).

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